The present application claims foreign priority under 35 U.S.C. xc2xa7119 of German Application No. 199 44 355.6 filed Sep. 16, 1999.
1. Field of the Invention
This invention concerns an optical arrangement in the beam path of a laser scanning microscope, with at least one adjustable spectrally sensitive element at the wavelength of the excitation light from a light source, which couples the excitation light from the light source into the microscope, at least partially blocks out excitation light scattered and reflected at an object, and does not block out the detection light coming from the object.
2. Description of the Related Art
Laser scanning microscopes of the generic type are known from the German Patent Application 199 06 757.0. AOTFs (Acousto-optical tunable filters) can be used, for example, as adjustable spectrally selective elements. They are generally birefringent crystals. Because of their birefringence, randomly polarized detection light is divided into two polarized partial beams, that is, into an ordinary and an extraordinary beam. The two beams are spatially separated after they pass through the crystal. Because of their pattern, the crystals do not have a cubic shape. Therefore polychromatic light is separated into its spectral components due to dispersion.
There are substantial problems in use of an adjustable spectrally selective element in the beam path of a laser scanning microscope because of the properties described above, as normally only a single non-separated detection beam is detected. The dispersed and divergent detection light must pass through an additional optical arrangement for detection to reduce the undesired effects of the adjustable spectrally selective element. Therefore it is necessary to follow the adjustable spectrally selective element by a second element of the same construction which makes the detection beam parallel after it passes through. Both those two elements are followed by a third optical element so that the parallel detection beam, comprising many detection partial beams, is converted to a convergent beam. Finally, a fourth element of the same design follows the three elements. It is able to reverse the dispersive and birefringent action of the first element. This arrangement is extremely tedious to adjust, is of substantial size, and is very expensive, primarily because of its use of four identical crystals.
This invention is based on the objective of designing and developing an optical arrangement in the beam path of a scanning laser microscope so as to allow simplification of the design of the long-known arrangement, and so to reduce the cost and expand the variations for detection previously provided. Such microscopes are known to have a light source, at least one adjustable spectrally selective element adjustable to the wavelength of excitation light from said light source for coupling in the excitation light from the light source into the microscope and at least partly blocking the excitation light scattered and reflected at an object from the detection beam path while transmitting detection light coming from the object on the beam path.
This objective is attained by an optical arrangement characterized by the fact that the adjustable spectrally selective element is followed by another optical component such that, after passing through it, the dispersive and/or birefringent properties of the detection light are detectable.
It is first recognized, according to the invention, that because of the birefringent property of an adjustable spectrally selective element which is used, a randomly polarized detection beam is separated into two detection partial beams with mutually perpendicular polarizations andxe2x80x94assuming that there is a suitable detection devicexe2x80x94they can be examined with particular respect to their polarization properties. Thus it is possible to make a polarizing laser scanning microscope. Because the distance from the adjustable spectrally selective element to the detector cannot be arbitrarily small, because of the structure, and because the detection light diverges after passing through the adjustable spectrally selective element, it has been possible to detect the light only with an overly large detector. The detector still had to have sufficient sensitivity with the same signal/noise ratio, because the total intensity per unit area of the detection light, already weak, is further reduced when distributed over a larger area. Therefore, another subsequent optical component is needed for effective detection. This component particularly converts the divergent light to non-divergent light so that it can be detected with detectors of ordinary size and sensitivity. It is also possible, in a manner according to the invention, to provide detection with respect to the dispersive properties of the detection light after passage through the element and the component.
An AOTF (acousto-optically tunable filter), an AOM (acousto-optical modulator) or an AOD (acousto-optical deflector) can be used as the adjustable spectrally selective element and as the other optical component. A birefringent crystal, a prism, or a lens could serve as the further optical component. A combination of the various optical components is also conceivable. For instance, an AOTF could be used as an adjustable spectrally selective element which is combined with a birefringent crystal as the optical component following the element.
It is very advantageous, for further processing of the detection light, divided into partial beams by the element, for the component to be placed after the element so that the light beams of the detection light emerge as nearly parallel as possible after passing through the component. If two elements of the same structure are used, that can be accomplished, for instance, with a mutually point-symmetric arrangement of the two components so as to take into consideration the particular form of component used in general. It is advantageous for the detection light to occur as parallel light beams after passing through the element and the component so that, as is usual in a conventional microscope, an infinite detection beam path is produced.
Depending on the actual applicational requirements for the laser scanning microscope to be made, it can be advantageous to detect a partial detection beam. Likewise, two or more detection partial beams can be detected simultaneously.
Multiple detection partial beams can be detected with separate detectors, with the detection partial beams preferably detected simultaneously. For practical accomplishment of the detection of multiple detection partial beams with a least one detector, it is advantageous to place at least one beam diverter in an appropriate detection partial beam path and arrange it so that the corresponding detection partial beam is directed to the detector assigned to it. The corresponding beam diverter is assigned to the element and the component so that the detection beam, which passes through the element and the component and is as nearly parallel as possible can be directed to the corresponding detector in unaltered beam form, because this is, due to the construction, for example, placed farther away from the element and the optical component.
With respect to an actual embodiment, the beam diverter could be made movable so that it could be placed in each of the detection partial beam paths. For example, if there were two detection partial beams, either one or the other detection partial beam could be detected with only one detector, depending on the instantaneous position of the movably mounted beam diverter. Quite generally, any detection partial beam could be detected in this manner by one detector. That is particularly advantageous if the laser scanning microscope is to have a very expensive detector selected for its detection characteristics, which is, for example, particularly low in noise, or is very sensitive in a particular wavelength region.
The detected detection partial beam can be a polarized ordinary beam of the detection light. Alternatively, it is also possible to detect the polarized extraordinary beam of the detection light. In one preferred embodiment, both the polarized partial beams are detected simultaneously with two separate detectors. This process gives a laser scanning microscope which can detect the detection light with respect to its polarization properties without inserting components such as a polarizer and analyzer, usually required, into the microscope beam path.
It is also possible, advantageously, to detect the properties of the detection light with a multiband detector or with a spectrometer. A multiband detector is known from German Patent DE 43 30 347 C2. Its content is assumed known, and is expressly included here. The dispersion of the detection light produced by the adjustable spectrally selective element can be utilized so that the multiband detector or spectrometer following the element and the optical component considers and furthermore intensifies the dispersion already partially accomplished. The spectrometer can be either a grating or prism spectrometer.
With respect to an actual embodiment, it can be advantageous for at least one detection partial beam to be blocked out of the detection beam path by a blocking means. A beam trap such as is commonly used in optical systems can serve as the blocking means. The blocking means could be movably mounted so that it can act intentionally on one or multiple detection partial beams. The means for moving the movably mounted blocking means is preferably in a plane perpendicular to the detection beam path.
A beam deflector can be placed after the element and the component in a manner according to the invention so that the light coming from the object, after passing through the element and the component, is reflected back so that the detection light can be detected by at least one detector after its second passage through the component and the element. This optical arrangement, comprising the adjustable spectrally selective element, the other optical component, and the beam diverter, has an action identical to the arrangement known from the state of the art, comprising four identical crystals placed in succession. Both arrangements can recombine the divergent and dispersively spread out detection light split up by the adjustable spectrally selective element into multiple partial beams back into a single coaxial detection beam. The design suggested here is, therefore, of considerable advantage, because only two instead of the four crystals are needed, the total size of the arrangement is reduced, adjustment work is reduced, and the cost can be reduced.
The beam diverter can be formed by an at least partially reflectorized rear side of the optical component. This can further reduce the number of components in the optical arrangement as well as the degrees of freedom in the adjustment.
With respect to an actual embodiment, the beam diverter is arranged and positioned so that the detection beam path running toward the beam diverter is at least slightly tilted in relation to the beam path running back from the beam diverter. It is advantageous to select the angle of tilt so that it is greater than 0 and smaller than 20 degrees. Then the detection light, after a double passage through the element and the component has a spatial beam displacement with respect to the detection light from the microscope so that the detection light reflected from the beam diverter can be directed from at least one further beam diverter to at least one detector.
The beam diverter following the element and the component is advantageously designed such that only one specifiable number of the detection partial beams coming from the object is reflected back into the component and the element. In this way, the beam diverter does not act on all the detection partial beams, so that, in an advantageous manner, only one specifiable portion of the detection light passes twice through the element and the component and so can be detected with appropriate detectors as a filtered coaxially recombined light beam.
In one preferred embodiment, both the detector beams emerging from the element and the component, as well as the detection partial beams reflected back into the component and the element are detected simultaneously with one detector. In particular, the detection partial beams reflected back into the component and the element can be detected as a recombined detection beam with a detector after passing through the component and the element. The detection partial beams emerging from the element and the component can likewise be detected simultaneously with the appropriate number of detector. In this case it is of considerable advantage for the beam diverter following the element and the component to be provided with at least one passage through which one or more detection partial beams leaving the element and component can pass out, while all the other detection partial beams are reflected back again into the component and the element. This beam diverter, with at least one passage, could be movable so that, for instance, only the polarized ordinary detection partial beam can pass through the beam diverter at one appropriate position of the beam diverter. The direction of movement of the movably arranged beam diverter is preferably in a plane perpendicular to the detection beam path.
A corresponding detection partial beam passing through the beam deflector could be detected by a detector. For that purpose, the corresponding detector could be combined directly with the beam diverter having a passage, so that no other optical components are needed. It is also conceivable that a detector measures a detection partial beam reflected back into the component and into the element.
The detection light can advantageously be detected after either single or double passage through the element and the component by removing from the beam path, or placing in the beam path, a movably mounted beam diverter which acts on all the detection partial beams and which is placed to follow the element and the component. In this way it is possible to switch, with very simple means, between two detector variants according to the invention.
Ordinary mirrors which reflect the detection partial beams, or the detection beam path, in the desired direction could be used as beam diverters. A phase-conjugate mirror would also be conceivable as a beam diverter. Aside from diverting the light, it would also be able to compensate or reverse distortions of the wave fronts of the detection light.